Part:BBa_K3040118
FadD
FadD is one of a regulatory enzyme in fatty acid biosynthesis in E. coli. , which is in charge of acetylating the fatty acid and produce a molecule, acetylated-fatty acid, that initiates promoter, pFadBA, to initiate the whole gene expression. We obtain the sequence of FadD directly by cell disruption of E. coli MG1655. After obtaining FadR, we further combine it with the promoter R0010 and a strong RBS B0030.
Characterization of fadD & fadL by BUCT 2021
Characterization of fadD & fadL:
Our project wants to enhance the decomposition of fatty acids by beta oxidation in intestinal tract, so we need to enhance the involved in translocation of long-chain fatty acids across the outer membrane. This composite part consists of lac promoter and two genes which are FadL and FadD. The FadL can help long-chain fatty acids involved in translocation across the outer membrane. FadL may form a specific channel. FadD catalyzes the esterification, concomitant with transport, of exogenous long-chain fatty acids into metabolically active CoA thioesters for subsequent degradation or incorporation into phospholipids. FadD is involved in the aerobic beta-oxidative degradation of fatty acids, which allows aerobic growth of E. coli on fatty acids as a sole carbon and energy source.
Construction:
PCR amplification technique was used to obtain the endogenous fadL and fadD of E. coli, then cut the two genes from the double strands. We use EcoRⅠand HindⅢ to cut the FadD, using BamHⅠand Bpu10Ⅰto cut the FadL. Last, we link the FadL and FadD by enzyme ligation method.
Experimental Result:
We configure the Gas Chromatograph test group: 200µl fermentation broth (obtain 20g/l palm oil), 2000µl petroleum ether-diethyl ether, 100µl methanol-KOH. From the chart, we can see the amount of palm oil is decreasing, and it proves this composite part can enhance the decomposition of fatty acids by beta oxidation in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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